1. Field of the Invention
The present invention pertains to a method for designing inhibitors of enzyme human immunodeficiency virus type 1 reverse transcriptase. More particularly, the method comprises the steps of (a) providing a three dimensional model of the receptor site in the prepolymerization complex of the p66 subunit of enzyme human immunodeficiency virus type 1 reverse transcriptase and a known nonnucleoside inhibitor; (b) locating the conserved residues in the p66 subunit which constitute the nonnucleoside inhibitor binding pocket; and (c) designing a new nonnucleoside inhibitor which possesses complementary structural features and binding forces to the residues in the p66 subunit nonnucleoside inhibitor binding pocket.
2. Description of the Background
The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference. For convenience, the disclosures are referenced in the following text and respectively grouped in the appended bibliography.
Acquired immunodeficiency syndrome (AIDS) is believed to be caused by the human immunodeficiency virus (HIV). Human immunodeficiency virus is a retrovirus which replicates in a human host cell. The human immunodeficiency virus appears to preferentially attack helper T-cells (T-lymphocytes or OKT4-bearing T-cells). When the helper T-cells are invaded by the virus, the T-cells become a human immunodeficiency virus producer. The helper T-cells are quickly destroyed causing the B-cells and other T-cells, normally stimulated by helper T-cells, to no longer function normally or produce sufficient lymphokines and antibodies to destroy the invading virus or other invading microbes.
Although the human immunodeficiency virus does not necessarily cause death, the virus generally causes the immune system to be so depressed that the human develops secondary infections such as herpes, cytomegalovirus, pneumocystis carinni, toxoplasmosis, tuberculosis, other mycobacteria, and other opportunistic infections. Kaposi's sarcoma, lymphomas, and cervical cancer may also occur. Some humans infected with the human immunodeficiency virus appear to live with little or no symptoms, but appear to have persistent infections, while others suffer mild immune system depression with symptoms such as weight loss, malaise, fever, and swollen lymph nodes. These syndromes have been called persistent generalized lymphadenopathy syndrome (PGL) and AIDS related complex (ARC) and generally develop into AIDS. Humans infected with the AIDS virus are believed to be persistently infective to others.
Human immunodeficiency virus is an extremely heterogeneous virus. The clinical significance of this heterogeneity is evidenced by the ability of the virus to evade immunological pressure, survive drug selective pressure, and adapt to a variety of cell types and growth conditions. A comparison of isolates among infected patients has revealed significant diversity, and within a given patient, changes in the predominant isolate over time have been noted and characterized. In fact, each patient infected with human immunodeficiency virus harbors a "quasispecies" of virus with a multitude of undetected viral variants present and capable of responding to a broad range of selective pressures, such as those imposed by the immune system or antiviral drug therapy. Therefore, diversity is a major obstacle to pharmacologic or immunologic control of human immunodeficiency virus infection. Human immunodeficiency virus infection has multiple mechanisms to maximize its potential for genetic heterogeneity. These mechanisms result in an extremely diverse population of virus capable of responding to a broad range of selective pressures, including the immune system and antiretroviral therapy, with the outgrowth of genetically altered virus.
When a patient with human immunodeficiency virus infection is initiated on antiretroviral therapy, there is generally a virologic response characterized by declining viremia and antigenemia. Unfortunately, the currently available antiretroviral agents which have undergone clinical evaluation have only limited benefit because most patients will ultimately have evidence of worsening disease and increasing viral burden. This progression often occurs in association with the emergence of drug-resistant human immunodeficiency virus. For example, most patients who are treated with 3'-azido-3'-deoxythymidine (AZT) will have initial evidence of improvement of clinical and laboratory parameters of human immunodeficiency virus infection. The duration of this benefit varies from patient to patient and is likely to be disease stage related. Ultimately, however, most patients will have progressive disease and genotypic or phenotypic evidence of the appearance of AZT-resistant human immunodeficiency virus. Since clinical failure and the appearance of virus with high level resistance to AZT both occur with evidence of increasing levels of viremia and changes in viral tropism, it has been difficult to ascribe the clinical failure solely to the development of AZT resistance. Nevertheless, it seems likely that AZT resistance ultimately contributes to the clinical failure seen in most patients receiving prolonged AZT therapy.
Enzyme human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is critical for the replication of HIV, which is the causative agent of acquired immunodeficiency syndrome (Goff, 1990). This enzyme (HIV-1 RT) plays a crucial role in the virus life cycle and is responsible for the conversion of the single-stranded RNA viral genome into double stranded DNA (Goff, 1990) (Furfine & Reardon, 1991). This DNA subsequently integrates into the host nucleus and through the normal metabolic pathway is able to produce progeny virus. Because of the distinct function of the reverse transcriptase in the virus life cycle, it is one of the most important targets in antiviral therapy. Two pharmacological classes of inhibitor molecules, i.e., nucleoside and nonnucleoside, have been found to be effective in halting the enzymatic function of the reverse transcriptase (Larder, 1993). Nucleoside inhibitors such as AZT (zidovudine, azidothymidine), ddC (Zalcitabine, 2', 3'-dideoxycytidine, Hivid), ddI (didanosine, 2', 3'-dideoxyinosine, Videx), and d4T (Stavudine, 2', 3'-didehydro-2', 3'-dideoxythymine) are chemically similar to the normal nucleosides and therefore can be converted to their triphosphate form and then used in the synthesis of DNA during reverse transcription. However, elongation of the DNA chain is blocked since these compounds lack a 3'-OH group which is essential for incorporation of additional nucleotides. Problems of cellular toxicity together with development of drug resistant variants of the virus have compromised the effective utility of these drugs. A number of pharmacologically active nonnucleoside inhibitors (NNI) have been identified. Many of these inhibitors appear highly potent, relatively nontoxic, and specifically inhibit HIV reverse transcriptase. However, the rapid emergence of HIV strains resistant to these compounds in vitro has become a major concern that may affect further development of these types of drugs (Larder, 1993). For example, nevirapine (BI-RG-587, 11-cyclopropyl-5, 11-dihydro-4-methyl-6H-dipyrido3,2-b:2',3'-e(1,4)diazepin-6-one), TIBO (Tetrahydroimidazo4,5,1-jk!1,4!benzodiazepin-2(1H)-one), HEPT (1-(2-hydroxyethoxymethyl)!-6-(phenylthio)thymine), BHAP (bis(heteroaryl)piperazine), and alpha-APA (alpha-anilinophenylacetamide) are highly studied compounds in this class (FIG. 3). Rapid mutations, in some cases within weeks or months, in the HIV-1 RT have been reported upon exposure of HIV-infected cells to these compounds. For example, mutations at Val108Ile, Tyr181Cys, and Tyr188His have been noted with pyridinone resistance, while Val1O6Ala, Tyr181Cys, and Tyr188Cys have been seen associated with nevirapine resistance (see Table 3).
Recently, the cocrystal structures of reverse transcriptase complexed with different nonnucleoside inhibitor molecules such as nevirapine, alpha-APA, HEPT, and, different derivatives of TIBO, have been determined by Arnold and colleagues (Ding et al., 1995A), (Ding et al., 1995B), and (Ren et al., 1995). All these crystallographic studies show that the chemically diverse class of nonnucleoside inhibitor molecules have common features of binding to reverse transcriptase.
Binding of these inhibitor compounds in reverse transcriptase is largely due to hydrophobic interactions. However, the contribution of individual amino acids in generating binding forces for these compounds is different in each case (Table 2). The shape of the hydrophobic pocket is generated by side chain arrangement of the residues, some of which are highly conserved in the reverse transcriptase class of enzymes and others whose mutations are known to develop resistance to the nonnucleoside class of inhibitor molecules. These observations are supported by several biochemical experiments (Balzarini et al., 1992) (Nunberg et al., 1992) (Schleif et al., 1992). Also, the structure of the unliganded structure (Rodgers et al., 1995) has indicated the rearrangement of positions and side chain conformations of certain amino acid residues which have created the cavity for nonnucleoside inhibitor binding. In the absence of a nonnucleoside inhibitor, there is no cavity in the binding region of the nonnucleoside inhibitor. The binding pocket is generated during the association of the nonnucleoside inhibitor. Drug resistance mutations obviously alter the shape of the nonnucleoside drug binding pocket resulting in the inability of the enzyme to bind a specific drug. Therefore, the identification and the availability of new molecules that will retain the binding specificity to this pocket in both the absence and presence of specific mutations is highly desired.
Many mutations reported to be associated with the nucleoside drug resistance of enzyme human immunodeficiency virus type 1 reverse transcriptase are clustered across the carboxylate triad in the p51 subunit of HIV-1 RT, but not in the catalytically active p66 subunit. This observation indicates some discrete role for the p51 subunit in the development of overall nucleoside drug resistance in HIV. Both nucleoside and nonnucleoside inhibitors have been shown to be quite effective in halting the propagation of HIV in tissue culture cells and in the animal model (Larder, B. A., 1993). A major problem with the continued use of these reverse transcriptase inhibitors has been the emergence of drug resistant mutant viruses (Larder, B. A., et al., 1989) (Kellam, R., et al., (1992). The problem of drug resistance in HIV is somewhat complicated since the resistance to an individual anti-RT drug does not always correlate in vitro with resistance of reverse transcriptase isolated from that strain (Larder, B. A., 1993) although a number of drug resistant HIV strains, isolated from patients on long term treatment, have indeed shown that viral resistance is due to mutations in the gene sequences coding for HIV-1 reverse transcriptase (Fitzgibbon, J. E., et al., (1993). The observed drug resistance has been well correlated to mutation at specific sites in HIV-1 RT. For example, mutations at Met 41, Asp 67, Lys 70, Thr 69, Lys 70, Leu 74, Met 184, Thr 215, and Lys 219 have been found to be associated with AZT and ddI resistance phenotype of HIV-1 (RT Lacey, S. F., et al., (1994) (Martin, J. L., et al., (1993) (St. Clair, M. H., et al., (1987). Recently, some additional mutation sites in HIV-1 RT were found in a strain of HIV that was isolated from a patient who was on a combination chemotherapy (AZT+ddI) for a period of one year (Shafer, R. W., et al., (1994).
An examination of residues conferring resistance to nucleoside inhibitors in the three-dimensional structure of HIV-1 RT showed that the mutation sites are dispersed over the entire finger subdomain of the p66 subunit of HIV-1 RT and that most of these sites are away from the catalytic center (FIG. 1) (Yadav, P. N. S., et al., 1995). The amino-acid side chains of many of these residues in the p66 subunit appear to be exposed to the outer surface, that is away from the cleft and towards the solvent medium, suggesting no direct participation of these residues in the polymerase function of the enzyme. The mechanism by which these mutations confer loss of recognition of nucleoside analogues (azido-or dideoxy-derivatives of dNTP) at the active site of HIV-1 RT has remained unexplained. To clarify the local environment around drug resistance sites in HIV-1 RT, a structural analysis of the regions in HIV-1 RT containing the mutations that confer nucleoside analogue resistance was undertaken. A model of the prepolymerization complex of HIV-1 RT template-primer and dNTP was used to analyze the location of the residues involved (FIG. 1). In contrast to the scattered distribution of mutant sites in the finger subdomain of the p66 subunit, these sites were found to be heavily concentrated in the vicinity of the carboxylate triad in the p51 subunit (FIG. 2).